Explanation of DNA Precipitation - (Sep/24/2006 )
Could someone explain the mechanism of DNA precipitation and the importance of the salt in the DNA solution before the Isopropanol precipitation? I understand that the Isopropanol precipitates out the DNA and the 70% Ethanol wash eliminates any salt that may have been precipitated out as well, but I'd like to understand more specifically the role of the salt in the first place and how this tecnique works.
i think salts help in aggregating DNA strands together by preventing repulsion induced by negatively-charged phosphate groups of the backbones..
by salts addition, you're creating a salted environment next DNA. Addition of alcohol is repulsive against DNA and the DNA starts aggregating. Salts are helping to create the agreagation both by helping "nucleation" step in precipitation, and by neutralizing the DNA charges.
The purpose of adding salts is to neutralize the charge on the sugar-phosphate backbone of the DNA. For a precipitation, you need to form ion pairs between the polyanion (DNA) and the cation (usually sodium or ammonium). The DNA and counterions are more or less in the free ion form rather than the ion pair form in dilute aqueous solution, and are surrounded by one or more layers of water molecules. Adding ethanol decreases the dielectric constant of the solution. The anions and cations form ion pairs and this results in precipitation of DNA.
Why it works... ("Frank, M. B. Ethanol Precipitation of Nucleic Acids. In: Frank, M. B. ed. Molecular Biology Protocols. (http://omrf.ouhsc.edu/~frank/etohppt.html). 1997. Oklahoma City. Revision Date: October 2, 1997." Notes that appear below were taken from a discussion on this topic from one of the bionet newsgroups.)
A discussion on this topic was posted to the "bionet.molbio.methds-reagnts" bulletin board in July 1992. A thorough discussion was presented by Scott Keeney, of the Biochemistry and Molecular Biology Department, University of California Berkeley (scottk@nuclease.Berkeley.edu). I have shortened the opening questions and extracted his answer from the newsgroup and present them below.
Q: What is the role of ethanol in precipitating DNA from aqueous solutions?
A: The purpose of adding salts is to neutralize the charge on the sugar-phosphate backbone of the DNA, but ethanol's task is a little more complex than "removing" the water. For a precipitation, you're interested in forming ion pairs between the polyanion (DNA) and the cation (Na+, Mg++, spermidine, protamine, etc). In dilute aqueous solution, DNA and counterions like Na+ and Mg++ are more or less in the free ion form rather than the ion pair form (that is, they are surrounded by one or more layers of water molecules).
Water has a high dielectric constant (e), which from Coulomb's Law tells us that the electrostatic force (F) between two ions of opposite charge is very low in water:
F = (Q1 * Q2)/(e * r2)
where Q is the charge on each ion and r is the distance between them. Adding organic solvent *decreases* the dielectric constant of the solution. As e goes down, F goes up and *BANG*, anion and cation form an ion pair and promptly swoon out of solution.
Q: Why does NaCl increase the stability of DNA duplexes, although you might expect salts to interfere with hydrogen bonds, rather than strengthen them?
A: The Na+ neutralizes the charge. Each strand of DNA has an enormous charge density (charge per unit volume), so the two strands tend to push each other apart. Cations added to the solution form a "cloud" of positive charges around the DNA. This cloud of counterions lowers the effective charge density and relieves the repulsion between the strands.
As for the effect of salt on hydrogen bonds, you have to realize that the hydrogen bonds formed between bases in duplex DNA contribute little to the stability of the duplex. For an interaction to stabilize the duplex, the interaction between bases must be stronger than the interaction of the bases with water (if bases are not paired with one another in a duplex, then they are surrounded by water). Hydrogen bonding between the amines, carbonyl oxygens, etc. of G-C or A-T is of the same energy (sometimes even less) than the hydrogen bonds these same groups would form with water if the DNA were single-stranded. (The H-bonds do contribute *something*: GC base pairs with three H-bonds are harder to melt than AT pairs with two.)
So, what drives DNA strands together? Entropy and enthalpy, of course. Entropy in the form of "hydrophobic" interactions between the bases (those big aromatic rings are quite hydrophobic, you know). Enthalpy in the form of favorable, stabilizing interactions between the pi electrons of the aromatic rings of bases as they stack on top of one another.
So, what do hydrogen bonds do, if they don't stabilize the duplex? They enforce the *specificity* of base pairing. Correct base pairing is nice, but doesn't add much. Incorrect base pairing, on the other hand, takes a lot away. Forcing unpaired H-bond donors and acceptors (i.e. hydrophilic groups) into a hydrophobic environment makes everybody unhappy.
“Ethanol or isopropanol or alcohols decrease the dielectric constant of the solution.
Water has a high dielectric constant (e), which from Coulomb's Law says that the electrostatic force (F) between two ions of opposite charge is very low in water:
F = (Q1 * Q2)/(e * r2)
where Q is the charge on each ion and r is the distance between them.
In an aqueous solution, DNA and counter ions like Na+ and Mg++ are more or less in the free ion form rather than the ion pair form i.e. they are surrounded by one or more layers of water molecules.
Adding organic solvent decreases the dielectric constant of the solution. As e goes down, F goes up and finally anion and cation form an ion pair and result in precipitation of DNA.” (http://micro.nwfsc.noaa.gov/forums/viewtopic.php?t=1623 s_atif_ali2002 (Malaysia))
it's more complicated....
just now know that the bases in DNA have a role in precipitation..